Single cell transcriptome reveals the molecular pathways of MADS-box genes in maize ovule development

Flowers are the reproductive structures of flowering plants, involved in seed production. A mature flower has four whorls of specialized organs that differentiate from heterogeneous cell clusters in the floral meristem. However, the mechanisms controlling the specialization of floral meristem cells remain elusive. Our early research identified a double mutant of two MADS-box genes (mads8;14) that lacks flower organs and repeatedly produces branch-like structures in the female inflorescence of maize (Zea mays L.). Here, single-cell transcriptomic analysis revealed that ZmMADS8/14 regulate ovule cell specialization and that branch-like structures are associated with defective floral meristem termination and enhanced proliferation of ovule epidermal cells in the mads8;14 mutant. ZmMADS8/14 restrict ZMM3 expression to the nucellus, whereas their loss results in ectopic ZMM3 expression co-localizing with KNOTTED1. ZMM3 suppresses the expression of floral organ identity genes such as B-class gene Zmm16/sterile tassel silky ear1, C-class gene Zea AGAMOUS1, E-class gene Bearded-ear1, and Drooping leaf1/Indeterminate floral apex1, while activating key factors involved in meristem maintenance. This indicates that ZmMADS8/14 suppress the meristematic activity but promote floral organ development by precisely regulating the level and domain of ZMM3 expression, leading to the formation of fertile florets and ovules. Additionally, knocking out ZMM3 partially rescues the defective phenotypes of the double mutant mads8;14, as well as increases the kernel number in the zmm3 mutant. These findings provide a new perspective for in-depth analysis of heterogeneous cell clusters, functions, and regulatory pathways in female floral organs, as well as potential targets for improving maize ear traits.